Journal: Cell reports

Article Title: Extracellular vesicles are carriers of adiponectin with insulin-sensitizing and anti-inflammatory properties.

doi: 10.1016/j.celrep.2023.112866

Figure Lengend Snippet: Figure 1. Adiponectin is the most enriched adipokine in VAT-derived EVs (A and B) Lean (Ctrl) and obese (Ob) VAT-derived lEV and sEV mode size comparison. Size-distribution curves (A) and mean mode size (B). Ctrl and Ob EV subtype size-distribution curves (A) are represented by plain or dashed lines, respectively, and are presented as the mean ± SEM (n = 4–6 independent EV preparations for each condition). (C and D) EV marker analysis from lean (Ctrl) or ob/ob (Ob) VAT explant-derived EV subpopulations. A significant enrichment in CD63 was observed for obese VAT-derived sEVs (D). (E) lEV and sEV secretion from VAT, SAT, and BAT explants. EV number secreted per gram of AT per 24 h is presented. (F) Increased lEV and sEV secretion from mouse VAT with obesity. EV secreted by total mouse VAT is presented. (G) EV secretion of human omental, mesenteric (Mesent.), and subcutaneous (Subcut.) AT collected from obese subjects. Secretion of EVs is presented as the number of EVs secreted per gram of human AT.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Adiponectin (murine) Thermofisher Scientific #PA1-054, RRID:AB_325789 Adiponectin (human) Abeomics #10-7597, RRID:AB_2943077 AKT pan (40D4) Cell signaling Cat #2920, RRID:AB_1147620 AKT pan polyclonal (in vivo studies) Cell signaling Cat #4691, RRID:AB_915783 Phospho-AKT (pAKT, Ser 473) Cell signaling Cat #4060S, RRID:AB_2315049 Alix BD Biosciences Cat #611620, RRID:AB_399062 Murine CD9 BD Pharmingen Cat #553758, RRID:AB_395032 human CD9 Santa Cruz Cat #SC-13118, RRID:AB_627213 Murine CD63 MBL Cat #D263-3, RRID:AB_1278815 Flotillin-2 BD Pharmingen Cat #610383, RRID:AB_397766 Mac2 Cedarlane Cat #CL8942AP, RRID:AB_10060357 MIF (human) R&D systems Cat #mab289, RRID:AB_2281975 Syntenin-1 Abcam Cat #ab19903, RRID:AB_445200 Zs-Green Clontech Cat #632598, RRID:AB_2943078 Goat anti-rat IgG2a Secondary antibody HRP conjugate (used for MaC2 staining) Invitrogen Cat# PA1-84709, RRID:AB_933942 Anti-adiponectin FITC coupled antibody Assay-Pro Cat #10361-05041, RRID:AB_2943079 IRDye 800CW and 680RD secondary antibody (anti-mouse and anti-rabbit) LI-COR Biosciences Cat #926–32211, RRID:AB_621843; Cat #926–32210,RRID:AB_621842; Cat #926–68071, RRID:AB_10956166; Cat #926-68070, RRID:AB_10956588 Chemicals, peptides, and recombinant proteins Bovine serum albumin (BSA) FFA free Sigma Cat #A7030 Collagenase A Roche Cat #10103586001 DAB OB Super Sensitive detection kit ImPath Cat #46538 DAB Quanto Thermofischer Scientific Cat #TA-125-QHDX Wash buffer for IHC ImPath Cat #45002 Antigen retrieval solution pH6.0 ImPath Cat #44998 Ultravision Hydrogen Peroxyde block Thermofisher Scientific Cat #TS-12-H2O2Q Goat serum Immunoreagents Cat #SP-004-VX10 IHC mounting medium CellPath Cat #SEA-1604-00A Qiazol Lysis reagent Qiagen Cat #79306 RNeasy mini kit Qiagen Cat #74104 SuperScriptII reverse Transcriptase Invitrogen Cat #18064-014 dNTP set 100mM Invitrogen Cat #10297-018 Random hexamers Invitrogen Cat #48190-011 QIAquick PCR purification kit Qiagen Cat #28106 RNaseOut Ribonuclease inhibitor Invitrogen Cat #10777-019 Maxima SYBR Green qPCR master mix 2X Thermofisher Scientific Cat #K0253 96-Well PCR plates Thermofisher Scientific Cat #AB0700 DMEM 4.5 g/L glucose Gibco Cat #41966-029 DMEM 1 g/L glucose Gibco Cat #31885-023 Elisa mouse Adiponectin/Acrp30 R&DSystems Cat #DY1119 and #DY008 (Continued on next page) 16 Cell Reports 42, 112866, August 29, 2023

Techniques: Derivative Assay, Comparison, Marker

Journal: Cell reports

Article Title: Extracellular vesicles are carriers of adiponectin with insulin-sensitizing and anti-inflammatory properties.

doi: 10.1016/j.celrep.2023.112866

Figure Lengend Snippet: Figure 3. Plasma EVs represent stable carriers of adiponectin (A) VAT-derived EVs are retrieved in the blood circulation, as illustrated by flow-cytometry detection of ZsGreen+ EVs in platelet-free plasma (PFP). Ctrl, control; AdipoZS1, Cre AdipoZs1 mice; AdipoZS1, Cre+ AdipoZs1 mice. (B) Adiponectinemia significantly decreases upon plasma EV removal in lean mice. (C) The presence of adiponectin was confirmed in mouse plasma circulating EVs. Plasma EV subtypes were isolated from lean and obese mice. Ten micrograms of each EV subtype was resolved by SDS-PAGE under reducing conditions (R) or nonreducing unheated conditions (NR). One representative blot (out of two experiments performed) is presented. (D) Adiponectin clearance measurement following the injection of serum or EV-depleted serum in adiponectin KO mice. n = 6 mice injected per group. (E) Western blot of human plasma sEVs confirms the decreased amount of adiponectin in sEVs isolated from obese patients compared to control patients. A representative blot (out of three independent experiments performed) is presented with quantification of the Adpn blot signal intensity.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Adiponectin (murine) Thermofisher Scientific #PA1-054, RRID:AB_325789 Adiponectin (human) Abeomics #10-7597, RRID:AB_2943077 AKT pan (40D4) Cell signaling Cat #2920, RRID:AB_1147620 AKT pan polyclonal (in vivo studies) Cell signaling Cat #4691, RRID:AB_915783 Phospho-AKT (pAKT, Ser 473) Cell signaling Cat #4060S, RRID:AB_2315049 Alix BD Biosciences Cat #611620, RRID:AB_399062 Murine CD9 BD Pharmingen Cat #553758, RRID:AB_395032 human CD9 Santa Cruz Cat #SC-13118, RRID:AB_627213 Murine CD63 MBL Cat #D263-3, RRID:AB_1278815 Flotillin-2 BD Pharmingen Cat #610383, RRID:AB_397766 Mac2 Cedarlane Cat #CL8942AP, RRID:AB_10060357 MIF (human) R&D systems Cat #mab289, RRID:AB_2281975 Syntenin-1 Abcam Cat #ab19903, RRID:AB_445200 Zs-Green Clontech Cat #632598, RRID:AB_2943078 Goat anti-rat IgG2a Secondary antibody HRP conjugate (used for MaC2 staining) Invitrogen Cat# PA1-84709, RRID:AB_933942 Anti-adiponectin FITC coupled antibody Assay-Pro Cat #10361-05041, RRID:AB_2943079 IRDye 800CW and 680RD secondary antibody (anti-mouse and anti-rabbit) LI-COR Biosciences Cat #926–32211, RRID:AB_621843; Cat #926–32210,RRID:AB_621842; Cat #926–68071, RRID:AB_10956166; Cat #926-68070, RRID:AB_10956588 Chemicals, peptides, and recombinant proteins Bovine serum albumin (BSA) FFA free Sigma Cat #A7030 Collagenase A Roche Cat #10103586001 DAB OB Super Sensitive detection kit ImPath Cat #46538 DAB Quanto Thermofischer Scientific Cat #TA-125-QHDX Wash buffer for IHC ImPath Cat #45002 Antigen retrieval solution pH6.0 ImPath Cat #44998 Ultravision Hydrogen Peroxyde block Thermofisher Scientific Cat #TS-12-H2O2Q Goat serum Immunoreagents Cat #SP-004-VX10 IHC mounting medium CellPath Cat #SEA-1604-00A Qiazol Lysis reagent Qiagen Cat #79306 RNeasy mini kit Qiagen Cat #74104 SuperScriptII reverse Transcriptase Invitrogen Cat #18064-014 dNTP set 100mM Invitrogen Cat #10297-018 Random hexamers Invitrogen Cat #48190-011 QIAquick PCR purification kit Qiagen Cat #28106 RNaseOut Ribonuclease inhibitor Invitrogen Cat #10777-019 Maxima SYBR Green qPCR master mix 2X Thermofisher Scientific Cat #K0253 96-Well PCR plates Thermofisher Scientific Cat #AB0700 DMEM 4.5 g/L glucose Gibco Cat #41966-029 DMEM 1 g/L glucose Gibco Cat #31885-023 Elisa mouse Adiponectin/Acrp30 R&DSystems Cat #DY1119 and #DY008 (Continued on next page) 16 Cell Reports 42, 112866, August 29, 2023

Techniques: Clinical Proteomics, Derivative Assay, Cytometry, Control, Isolation, SDS Page, Injection, Western Blot

Journal: Cell reports

Article Title: Extracellular vesicles are carriers of adiponectin with insulin-sensitizing and anti-inflammatory properties.

doi: 10.1016/j.celrep.2023.112866

Figure Lengend Snippet: Figure 4. EV-associated adiponectin maintains insulin sensitivity in target cells (A and B) Adiponectin-enriched sEVs reverse insulin resistance in hepatocytes. Ctrl sEVs and Ob sEVs correspond to VAT-derived sEVs isolated from either lean or obese VAT, respectively. Adpn-Ob sEVs refer to the enrichment of Ob sEVs with adiponectin (following their preincubation with EV-free conditioned media). Ins, insulin; Palm, palmitate. (C) Silencing of both AdipoR1 and AdipoR2 significantly reduces the VAT-derived sEV insulin-sensitizing effects. Scr, Scramble; R1, AdipoR1; R2, AdipoR2. (D) Rapid and time-dependent internalization of fluorescent adiponectin-Venus (Adpn-Venus) internalization in hepatocytes. Scale bars, 50 mm. (E) Adiponectin enrichment in sEVs is responsible for their insulin-sensitizing effects, independent of their sEV cellular origin. Dot plots represent independent experiments. Data are presented as the mean ± SEM. Statistical differences were assessed using one-way ANOVA. Statistical significancy is indicated for each panel as follows : *p % 0.05, **p % 0.01, ***p % 0.01,****p % 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Adiponectin (murine) Thermofisher Scientific #PA1-054, RRID:AB_325789 Adiponectin (human) Abeomics #10-7597, RRID:AB_2943077 AKT pan (40D4) Cell signaling Cat #2920, RRID:AB_1147620 AKT pan polyclonal (in vivo studies) Cell signaling Cat #4691, RRID:AB_915783 Phospho-AKT (pAKT, Ser 473) Cell signaling Cat #4060S, RRID:AB_2315049 Alix BD Biosciences Cat #611620, RRID:AB_399062 Murine CD9 BD Pharmingen Cat #553758, RRID:AB_395032 human CD9 Santa Cruz Cat #SC-13118, RRID:AB_627213 Murine CD63 MBL Cat #D263-3, RRID:AB_1278815 Flotillin-2 BD Pharmingen Cat #610383, RRID:AB_397766 Mac2 Cedarlane Cat #CL8942AP, RRID:AB_10060357 MIF (human) R&D systems Cat #mab289, RRID:AB_2281975 Syntenin-1 Abcam Cat #ab19903, RRID:AB_445200 Zs-Green Clontech Cat #632598, RRID:AB_2943078 Goat anti-rat IgG2a Secondary antibody HRP conjugate (used for MaC2 staining) Invitrogen Cat# PA1-84709, RRID:AB_933942 Anti-adiponectin FITC coupled antibody Assay-Pro Cat #10361-05041, RRID:AB_2943079 IRDye 800CW and 680RD secondary antibody (anti-mouse and anti-rabbit) LI-COR Biosciences Cat #926–32211, RRID:AB_621843; Cat #926–32210,RRID:AB_621842; Cat #926–68071, RRID:AB_10956166; Cat #926-68070, RRID:AB_10956588 Chemicals, peptides, and recombinant proteins Bovine serum albumin (BSA) FFA free Sigma Cat #A7030 Collagenase A Roche Cat #10103586001 DAB OB Super Sensitive detection kit ImPath Cat #46538 DAB Quanto Thermofischer Scientific Cat #TA-125-QHDX Wash buffer for IHC ImPath Cat #45002 Antigen retrieval solution pH6.0 ImPath Cat #44998 Ultravision Hydrogen Peroxyde block Thermofisher Scientific Cat #TS-12-H2O2Q Goat serum Immunoreagents Cat #SP-004-VX10 IHC mounting medium CellPath Cat #SEA-1604-00A Qiazol Lysis reagent Qiagen Cat #79306 RNeasy mini kit Qiagen Cat #74104 SuperScriptII reverse Transcriptase Invitrogen Cat #18064-014 dNTP set 100mM Invitrogen Cat #10297-018 Random hexamers Invitrogen Cat #48190-011 QIAquick PCR purification kit Qiagen Cat #28106 RNaseOut Ribonuclease inhibitor Invitrogen Cat #10777-019 Maxima SYBR Green qPCR master mix 2X Thermofisher Scientific Cat #K0253 96-Well PCR plates Thermofisher Scientific Cat #AB0700 DMEM 4.5 g/L glucose Gibco Cat #41966-029 DMEM 1 g/L glucose Gibco Cat #31885-023 Elisa mouse Adiponectin/Acrp30 R&DSystems Cat #DY1119 and #DY008 (Continued on next page) 16 Cell Reports 42, 112866, August 29, 2023

Techniques: Derivative Assay, Isolation

Journal: Cell reports

Article Title: Extracellular vesicles are carriers of adiponectin with insulin-sensitizing and anti-inflammatory properties.

doi: 10.1016/j.celrep.2023.112866

Figure Lengend Snippet: Figure 5. EV-associated adiponectin reverses HFD-induced insulin resistance in mice (A) NHS ester-labeled VAT-derived sEV organ biodistribution. Licor fluorescent tissue imaging is presented in Figure S4A. (B) Adoptive transfer of sEV-associated adiponectin limits weight gain induced by a high-fat diet (HFD). A standard diet (SD) is presented as a control to demonstrate HFD-induced weight gain compared to body weight on a regular chow diet. Number of animals per group: SD, n = 3; NaCl, n = 15; Ctrl sEVs, n = 13; AdpnKO sEVs, n = 11. (C) Tissue weights at sacrifice of mice that received i.p. injections of Ctrl sEVs, AdpnKO sEVs, or NaCl (vehicle) over the course of 5 weeks of HFD compared to mice maintained on an SD.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Adiponectin (murine) Thermofisher Scientific #PA1-054, RRID:AB_325789 Adiponectin (human) Abeomics #10-7597, RRID:AB_2943077 AKT pan (40D4) Cell signaling Cat #2920, RRID:AB_1147620 AKT pan polyclonal (in vivo studies) Cell signaling Cat #4691, RRID:AB_915783 Phospho-AKT (pAKT, Ser 473) Cell signaling Cat #4060S, RRID:AB_2315049 Alix BD Biosciences Cat #611620, RRID:AB_399062 Murine CD9 BD Pharmingen Cat #553758, RRID:AB_395032 human CD9 Santa Cruz Cat #SC-13118, RRID:AB_627213 Murine CD63 MBL Cat #D263-3, RRID:AB_1278815 Flotillin-2 BD Pharmingen Cat #610383, RRID:AB_397766 Mac2 Cedarlane Cat #CL8942AP, RRID:AB_10060357 MIF (human) R&D systems Cat #mab289, RRID:AB_2281975 Syntenin-1 Abcam Cat #ab19903, RRID:AB_445200 Zs-Green Clontech Cat #632598, RRID:AB_2943078 Goat anti-rat IgG2a Secondary antibody HRP conjugate (used for MaC2 staining) Invitrogen Cat# PA1-84709, RRID:AB_933942 Anti-adiponectin FITC coupled antibody Assay-Pro Cat #10361-05041, RRID:AB_2943079 IRDye 800CW and 680RD secondary antibody (anti-mouse and anti-rabbit) LI-COR Biosciences Cat #926–32211, RRID:AB_621843; Cat #926–32210,RRID:AB_621842; Cat #926–68071, RRID:AB_10956166; Cat #926-68070, RRID:AB_10956588 Chemicals, peptides, and recombinant proteins Bovine serum albumin (BSA) FFA free Sigma Cat #A7030 Collagenase A Roche Cat #10103586001 DAB OB Super Sensitive detection kit ImPath Cat #46538 DAB Quanto Thermofischer Scientific Cat #TA-125-QHDX Wash buffer for IHC ImPath Cat #45002 Antigen retrieval solution pH6.0 ImPath Cat #44998 Ultravision Hydrogen Peroxyde block Thermofisher Scientific Cat #TS-12-H2O2Q Goat serum Immunoreagents Cat #SP-004-VX10 IHC mounting medium CellPath Cat #SEA-1604-00A Qiazol Lysis reagent Qiagen Cat #79306 RNeasy mini kit Qiagen Cat #74104 SuperScriptII reverse Transcriptase Invitrogen Cat #18064-014 dNTP set 100mM Invitrogen Cat #10297-018 Random hexamers Invitrogen Cat #48190-011 QIAquick PCR purification kit Qiagen Cat #28106 RNaseOut Ribonuclease inhibitor Invitrogen Cat #10777-019 Maxima SYBR Green qPCR master mix 2X Thermofisher Scientific Cat #K0253 96-Well PCR plates Thermofisher Scientific Cat #AB0700 DMEM 4.5 g/L glucose Gibco Cat #41966-029 DMEM 1 g/L glucose Gibco Cat #31885-023 Elisa mouse Adiponectin/Acrp30 R&DSystems Cat #DY1119 and #DY008 (Continued on next page) 16 Cell Reports 42, 112866, August 29, 2023

Techniques: Labeling, Derivative Assay, Imaging, Adoptive Transfer Assay, Control

Journal: Cell reports

Article Title: Extracellular vesicles are carriers of adiponectin with insulin-sensitizing and anti-inflammatory properties.

doi: 10.1016/j.celrep.2023.112866

Figure Lengend Snippet: Figure 6. EV-associated adiponectin dis- plays anti-inflammatory properties (A) Macrophage (Mac2) staining in SAT (upper panels) and VAT (lower panels) depots from sEV-injected mice reveal the anti-inflammatory properties of Ctrl sEVs treatment. HFD-induced adipocyte hypertrophy and HFD-related macro- phage infiltration are observed in both SAT and VAT compared to SD-fed conditions. Scale bars, 50 mm. (B and C) qPCR mRNA expression of inflammatory markers in VAT (B) and liver (C) tissues. Dot plots represent the number of independent animals analyzed. Data are presented as the mean ± SEM. Statistical differences were calculated compared to the HFD + NaCl group using one-way ANOVA followed by the Kruskal-Wallis test. Statistical significancy is indicated for each panel as follows : *p % 0.05, **p % 0.01, ***p % 0.01,****p % 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Adiponectin (murine) Thermofisher Scientific #PA1-054, RRID:AB_325789 Adiponectin (human) Abeomics #10-7597, RRID:AB_2943077 AKT pan (40D4) Cell signaling Cat #2920, RRID:AB_1147620 AKT pan polyclonal (in vivo studies) Cell signaling Cat #4691, RRID:AB_915783 Phospho-AKT (pAKT, Ser 473) Cell signaling Cat #4060S, RRID:AB_2315049 Alix BD Biosciences Cat #611620, RRID:AB_399062 Murine CD9 BD Pharmingen Cat #553758, RRID:AB_395032 human CD9 Santa Cruz Cat #SC-13118, RRID:AB_627213 Murine CD63 MBL Cat #D263-3, RRID:AB_1278815 Flotillin-2 BD Pharmingen Cat #610383, RRID:AB_397766 Mac2 Cedarlane Cat #CL8942AP, RRID:AB_10060357 MIF (human) R&D systems Cat #mab289, RRID:AB_2281975 Syntenin-1 Abcam Cat #ab19903, RRID:AB_445200 Zs-Green Clontech Cat #632598, RRID:AB_2943078 Goat anti-rat IgG2a Secondary antibody HRP conjugate (used for MaC2 staining) Invitrogen Cat# PA1-84709, RRID:AB_933942 Anti-adiponectin FITC coupled antibody Assay-Pro Cat #10361-05041, RRID:AB_2943079 IRDye 800CW and 680RD secondary antibody (anti-mouse and anti-rabbit) LI-COR Biosciences Cat #926–32211, RRID:AB_621843; Cat #926–32210,RRID:AB_621842; Cat #926–68071, RRID:AB_10956166; Cat #926-68070, RRID:AB_10956588 Chemicals, peptides, and recombinant proteins Bovine serum albumin (BSA) FFA free Sigma Cat #A7030 Collagenase A Roche Cat #10103586001 DAB OB Super Sensitive detection kit ImPath Cat #46538 DAB Quanto Thermofischer Scientific Cat #TA-125-QHDX Wash buffer for IHC ImPath Cat #45002 Antigen retrieval solution pH6.0 ImPath Cat #44998 Ultravision Hydrogen Peroxyde block Thermofisher Scientific Cat #TS-12-H2O2Q Goat serum Immunoreagents Cat #SP-004-VX10 IHC mounting medium CellPath Cat #SEA-1604-00A Qiazol Lysis reagent Qiagen Cat #79306 RNeasy mini kit Qiagen Cat #74104 SuperScriptII reverse Transcriptase Invitrogen Cat #18064-014 dNTP set 100mM Invitrogen Cat #10297-018 Random hexamers Invitrogen Cat #48190-011 QIAquick PCR purification kit Qiagen Cat #28106 RNaseOut Ribonuclease inhibitor Invitrogen Cat #10777-019 Maxima SYBR Green qPCR master mix 2X Thermofisher Scientific Cat #K0253 96-Well PCR plates Thermofisher Scientific Cat #AB0700 DMEM 4.5 g/L glucose Gibco Cat #41966-029 DMEM 1 g/L glucose Gibco Cat #31885-023 Elisa mouse Adiponectin/Acrp30 R&DSystems Cat #DY1119 and #DY008 (Continued on next page) 16 Cell Reports 42, 112866, August 29, 2023

Techniques: Staining, Injection, Expressing

Journal: iScience

Article Title: circIFNGR2 regulating ankylosing spondylitis-associated inflammation through macrophage polarization

doi: 10.1016/j.isci.2023.107325

Figure Lengend Snippet:

Article Snippet: On day 21 from the first immunization, boost injection of 100 μL bovine type II collagen emulsified in incomplete Freund’s adjuvant (Chondrex, WA, USA) was conducted.

Techniques: Virus, Recombinant, Adjuvant, In Vivo, SYBR Green Assay, Transfection, Lysis, Blocking Assay, Western Blot, DNA Extraction, Protein Extraction, Enzyme-linked Immunosorbent Assay, Purification, RNA Immunoprecipitation, Luciferase, Software